1. Technical Field
The field of the subject invention is the generation and use of major histocompatibility complex antigen lacking cells and organs lacking expression of functional major histocompatibility complex (MHC) antigen which may serve as universal donors in cellular and organ therapies including transplantation and to produce chimeric non-human mammals.
2. Background
To protect vertebrates from disease and infection, elaborate protective systems have evolved. In mammals, the immune system serves as the primary defense with many different types of cells and mechanisms to protect the host. A wide variety of hematopoietic cells exists, with the major protective lineages being lymphoid and myeloid.
The immune system, which results from cells of the lymphoid and myeloid lineages is developed in vivo, so as to recognize self from non-self. Those aberrant situations where the immune system attacks self, such as rheumatoid arthritis, lupus erythematosus, and certain forms of diabetes, are evidence of the importance to the host that only foreign agents be attacked. The protective mechanism which protects the host from disease, as a result of invasion of viruses, bacteria, or other pathogens, is also able to recognize cells which come from a different mammalian host, even an allogeneic host.
As part of the system for the self-versus-non-self recognition, the surface membrane protein major histocompatibility complex (MHC) antigens serve an important role. Each host has a personal set of Class I and II MHC antigens, which serve to distinguish that host from other hosts. The T-lymphoid system is predicated upon recognition of the presence of such MHC antigens as self. Where transplantation from another allogeneic host occurs, unless the transplant is matched with the host or the host is immunocompromised, the transplant may be attacked and destroyed by the immune system. When a transplant occurs which includes lymphocytes, monocytes or progenitors thereof, particularly bone marrow, a graft may attack the host as foreign, resulting in graft-versus-host disease.
There are many situations where one may wish to transplant cells into a recipient host where the recipient""s cells are missing, damaged or dysfunctional. When the host is immunocompromised, there may be an interest in transfusing specific white cells, particularly T-cells, which may protect the host from various diseases. When the host lacks the ability to raise a defense against a particular disease, there may also be an interest in administering specific T-cells or B-cells or precursors thereof which may supplement the host""s compromised immune system. In other cases, where certain cells are lacking, such as islets of Langerhans in the case of diabetes, or cells which secrete dopamine in the case of Parkinson""s disease, or bone marrow cells in various hematopoietic diseases, or muscle cells in muscle wasting disease, or retinal epithelial cells in visual disorders, or keratinocytes for burns and non-healing wounds, it would be desirable to be able to provide cells which could fulfill the desired function. In order for the cells to be effective, they must be safe from attack by the host, so that they may function without being destroyed by the immune system. It is therefore of interest to find effective ways to produce cells which may function, proliferate, and differentiate as appropriate, while being safe from attack by a recipient""s immune system, for example by the use of gene targeting to inactivate the expression of gene products that cause rejection of the transplanted cells. The same reasons apply to the use of organs for transplantation including but not limited to the heart, lung, liver and kidney.
Homologous recombination permits site-specific modifications in endogenous genes and thus inherited or acquired mutations may be corrected, and/or novel alterations may be engineered into the genome. The application of homologous recombination to gene therapy depends on the ability to carry out homologous recombination efficiently in normal diploid somatic cells. Homologous recombination or xe2x80x9cgene targetingxe2x80x9d in normal, somatic cells for transplantation represents a potentially powerful method for gene therapy; however, with the exception of pluripotent mouse embryonic stem (ES) cells, and continuous cell lines, homologous recombination has not been reported for a well-characterized, non-transformed, i.e., xe2x80x9cnormalxe2x80x9d mammalian somatic cell. In contrast to mouse ES cell lines, normal somatic human cells may have a finite life span in vitro (Hayflick and Moorhead, Exptl. Cell. Res. 25:585-621 (1961)). This makes their modification by gene targeting especially challenging, given the low efficiency of this process, i.e., 10xe2x88x925 to 10xe2x88x928 recombinants/input cell. Moreover, this process is further complicated by the fact that mammalian cells tend to integrate transfected DNA at random sites 100 to 1000 fold more efficiently than at the homologous site.
The present invention discloses methods for targeting non-transformed diploid somatic cells to inactivate genes associated with MHC antigen expression, including the xcex22-Microglobulin and IFN-xcex3R genes in cells such as retinal epithelial cells, keratinocytes and myoblasts. These methods provide novel targeting means for inactivating target genes resulting in lack of expression of functional MHC. In a method of the invention for targeting integral membrane proteins, the role of such proteins may be studied, and their expression manipulated, for example membrane proteins that serve as receptors, such as T cell receptors.
There is also substantial interest in being able to study various physiological processes in vivo in an animal model. In many of these situations, one would wish to have a specific gene(s) inactivated or introduced in a site-directed fashion. Where all or a substantial proportion of the cells present in the host would be mutated, the various processes could be studied. In addition, heterozygous hosts having one wild-type gene and one mutated gene could be mated to obtain homozygous hosts, so that all of the cells would have the appropriate modification. Such genetically mutated animals could serve for screening drugs, investigating physiologic processes, developing new products, and the like.
A number of papers describe the use of homologous recombination in mammalian cells, including human cells. Illustrative of these papers are Kucherlapati et al., Proc. Natl. Acad. Sci. USA 81:3153-3157, 1984; Kucherlapati et al., Mol. Cell. Bio. 5:714-720, 1985; Smithies et al, Nature 317:230-234, 1985; Wake et al., Mol. Cell. Bio. 8:2080-2089, 1985; Ayares et al., Genetics 111:375-388, 1985; Ayares et al., Mol. Cell. Bio. 7:1656-1662, 1986; Song et al., Proc. Natl. Acad. Sci. USA 84:6820-6824, 1987; Thomas et al. Cell 44:419-428, 1986; Thomas and Capecchi, Cell 51: 503-512, 1987; Nandi et al., Proc. Natl. Acad. Sci. USA 85:3845-3849, 1988; and Mansour et al., Nature 336:348-352, 1988.
Evans and Kaufman, Nature 294:146-154, 1981; Doetschman et al., Nature 330:576-578, 1987; Thoma and Capecchi, Cell 51:503-512,4987; Thompson et al., Cell 56:316-321, 1989; individually describe various aspects of using homologous recombination to create specific genetic mutations in embryonic stem cells and to transfer these mutations to the germline. The polymerase chain reaction used for screening homologous recombination events is described in Kim and Smithies, Nucleic Acids Res. 16:8887-8903, 1988; and Joyner et al., Nature 338:153-156, 1989. The combination of a mutant polyoma enhancer and a thymidine kinase promoter to drive the neomycin gene has been shown to be active in both embryonic stem cells and EC cells by Thomas and Capecchi, supra, 1987; Nicholas and Berg (1983) in Teratocarcinoma Stem Cell, eds. Siver, Martin and Strikland (Cold Spring Harbor Lab., Cold Spring Harbor, N.Y. (pp. 469-497); and Linney and Donerly, Cell 35:693-699, 1983.
Bare lymphocytes are described in Schuurman et al., The Thymocyte in xe2x80x9cBare Lymphocytexe2x80x9d Syndrome In: Microenvironments in the Lymphoid System, ed. Klaus, G. G. B. Plenum Press, NY, pp. 921-928 (1985); Sullivan et al., J. Clin. Invest. 76;:75-79 (1985); Lisowska-Grospierre et al., ibid. 76:381-385 (1985); Arens, et al., J. Inf. Dis. 156:837-841 (1987); Clement et al. J. Clin. Invest. 81:669-675 (1988); Sugiyama et al., Chest 89:398-401 (1986); and Hume et al., Human Immnology 25:1-11 (1989).
Transplantation of various normal somatic cells to treat hereditary disease has been reported (Blaese et al., Human Gene Ther. 4:521-527 (1993)). Recent transplantation experiments suggest that myoblast transplantation represents a potentially useful vehicle for drug delivery (Barr et al., Science 254:1507-1509 (1991) and Dhawan et al., Science 254:1509-1512 (1991)). For example, transplantation of normal myoblasts to treat Duchenne muscular dystrophy and other muscle degeneration and wasting diseases has been proposed by Partridge, Muscle and Nerve 14:197-212 (1991).
Interferon-gamma (IFN-xcex3) is a cytokine that is produced during the process of infection and inflammation which exhibits potential antiviral, anti-proliferative and immunomodulatory effects (Trinchieri et al., Immunol. Today 6:131.136. (1985); Pestka et al., Ann. Rev. Biochem. 56:727-777 (1987); and Farrar et al., Ann. Rev. Immunol. 11:571-611 (1993)). Many of these actions are thought to be mediated by binding to a ubiquitously expressed, high affinity cell surface receptor, the IFN-xcex3 receptor, (Aguet et al., Cell 55:273-280 (1988)) which triggers the induction of MHC antigens (Rosa et al., Immunol. Today 5:261-262 (1984)). Because IFN-xcex3 upregulates the expression of the products of the genes encoding xcex22-microglobulin and the transporter of the antigenic peptides TAP-1 and TAP-2 associated with expression of MHC Class I complex (Germain et al., Ann. Rev. Immunol. 11:403-450 (1993)), as well as expression of MHC Class I and II molecules Pestka et al., Ann. Rev. Biochem. 56:727-777 (1987); Farrar et al., Ann. Rev. Immunol. 11:571-611 (1993); Rosa et al., Immunol. Today 5:262-262 (1984) and Trowsdale et al., Nature 348:741-744 (1990)), blocking the effects of IFN-xcex3 by inactivating its receptor using homologous recombination may decrease cellular rejection of allogeneic transplants. Cultured human myoblasts express both MHC Class I and Class 11 antigens at very low levels, but their expression increases significantly after treatment with INF-xcex3 (Bao et al., Immunol. Cell Biol. 68:235-242 (1990)). Thus, in an allogeneic recipient, INF-xcex3 released by T cells infiltrating the transplantation site may upregulate MHC expression, resulting in rejection of the donor myoblasts. As the expression of MHC Class I can also be upregulated by other cytokines such as INF-xcex1 and INF-xcex2 and IL-1, inactivation of the INF-xcex3R may be combined with inactivation of other genes important for MHC Class I expression, for example, IL-lR, TAP 1 and/or TAP 2 and/or xcex22-microglobulin and/or proteasome genes, to produce universal donor myoblasts that may be transplanted across histocompatibility barriers.
Schwartzberg et at. Proc. Natl. Acad. Sci. USA 87:3210-3214 (1990) describe a targeted gene disruption of an endogenous c-abl locus by homologous recombination with DNA encoding a selectable fusion protein. Other references of interest include Jasin et al., Genes and Development 4:157-166 (1990) describing gene targeting at the human CD4 locus by epitope addition and Doetschman et al., Proc. Natl. Acad. Sci. (USA) 85:8583-8587 (1988) which describe targeted mutation of the Hprt gene in mouse embryonic stem cells using a targeting DNA fragment containing a promoterless neo gene. Other references describing various uses of the Neo gene in targeting include Sedivy and Sharp, Proc. Natl. Acad. Sci. (USA) 86:227-231 (1989); Riele et al, Letters to Nature 348:649-651 (1990); Jeannotte et al., Molec. and Cell. Biol. 11(11):5578-5585 (1991); Charron et al., Molec. Cell. Biol. 10 (4):1799-1804 (1990); Stanton et al., Molec. Cel. Biol. 10(12):6755-6758 (1990).
The successful application of gene targeting to somatic cell gene therapy requires the precise integration of exogenous DNA into the target locus without inducing other genetic alterations resulting in phenotypic abnormalities in the target cell. There exists an ongoing need for methods which enrich for the lower frequency recombinant events that occur in somatic cells as compared to the frequency of random recombination.
Mammalian cells lacking at least one functional major histocompatibility complex (MHC) antigen are provided which may serve to diminish immune attack when used for transplantation, particularly as universal donor cells, including non-transformed diploid human somatic cells, or as embryonic stem cells which may be used to produce chimeric mammals carrying the mutation. The cells are obtained as a result of homologous recombination. Particularly, by inactivating at least one allele of at least one MHC antigen chain, e.g., a MHC xcex1 chain, or xcex22-microglobulin, cells can be produced which have reduced capability for expression of functional MHC antigens. The resulting cells lacking functional MHC antigen may be used as donors for transplantation lacking markers for host (recipient) immune attack. The cells may be used to produce tissue for transplantation. The cells may also be used in vitro to interact with other cells. Transgenic mammals carrying this trait may be used in the study of immunodeficiency and may be used as a source of tissues and cells for transplantation.
Alternatively, cells containing inactivated genes associated with the expression of MHC antigen, for example, the INF-xcex3R gene, are obtained using the methods of the invention, to prevent the upregulation of MHC antigen expression in response to IFN-xcex3, resulting in the generation of donor cells deficient in the ability to upregulate expression of MHC antigen.
Methods and targeting constructs are provided wherein low frequency homologous recombination in non-transformed somatic cells may be rapidly detected. In a method of the invention, a DNA construct containing a strong promoter, an epitope that binds to a ligand for detection, and a selectable marker gene, is targeted to a sequence in the chromosome of a cell encoding the target locus for homologous recombination. When this DNA construct is transfected into cells, a fusion protein is expressed and-secreted outside of the cell. Additionally, novel methods and targeting constructs are provided for inactivation of integral membrane proteins by inserting a selectable marker gene into the protein coding region downstream from a sequence encoding a leader sequence and a transmembrane sequence. The targeting construct inserts the selectable maker gene into the gene encoding the integral membrane protein so as to be in reading frame with the upstream sequence and to encode a fusion protein with the functional marker on the cytoplasmic side of the membrane. Cells are transformed with the constructs using the methods of the invention and are selected by means of the selectable marker and then screened for the presence of recombinants.
Another method of the invention is for determining the effectiveness of a therapeutic agent to prevent transplant rejection in a mammal by administering a therapeutic agent, such as cyclosporine, to the mammal into which tissues or cells that lack expression of functional MHC antigen have been transplanted, and observing for the presence or absence of rejection of the transplanted tissues or cells over time.